Early approaches to chemical detection of analytes relied on chemical indicators to produce color changes or precipitation products giving readouts that are visible to the naked eye. These tests were therefore convenient, portable and economical, but relatively insensitive. More recently, the limits of detection have been dramatically lowered, both by the development of methods of separation to purify and concentrate the analyte and by the development of increasingly sophisticated instrumentation to detect analytes with greater specificity. However, both of these approaches require specialized equipment and skilled personnel to operate and are generally inapplicable to field testing.
There are many instances, such as in environmental studies, biomedical research and medical diagnosis, where there is a need for detection technology that can rapidly detect very low amounts of analytes as well as reduce the time for sample preparation, laboratory infrastructure and turnaround. For example, in localities where harsh socioeconomic conditions persist, diagnosis of infectious diseases would be benefited by techniques that are adaptable outside of conventional clinical laboratories. In addition, many military and commercial settings require the tracking of materials and/or documents and certification of their origin and authenticity. Tracking of materials or personnel may be accomplished by using a substance acting as a tag that is invisible until detected using specified analytical techniques. Presently, the methods of detecting such taggants are costly, laboratory-based and time-consuming.
The present invention provides assay compositions and methods that are adaptable to field use, simple to carry out, and able to detect very small amounts of analyte. The invention relies on amplification of a signal provided by the analyte by permitting the analyte directly or indirectly to trigger an autocatalytic chain reaction that amplifies the signal to produce products that are detectable by the naked eye.
Others have attempted to employ autocatalytic chain reactions (ACR's) to amplify analyte signals. For example, Dirks, et al., U.S. 2005/0260635 describe a method whereby metastable nucleic acid monomers can be made to self-assemble upon exposure to an analyte, thus obtaining a linear duplex polymer that can be detected by gel electrophoresis. No suggestion of a visible signal, such as color or fluorescence, is disclosed. Detection by fluorescence is, however, disclosed in Pierce, et al., U.S. 2006/0228733, a published application by the same group that employs a similar method of polymerization by an initiating oligonucleotide analyte. Similarly, in another publication of the same group, Pierce, et al., U.S. 2006/0234261, nanogold particles are used to detect the self-assembled polymers.
Zhang, et al., U.S. 2005/0227259 describe generation of an amplified signal indicative of a target nucleic acid molecule using detection by fluorescent resonance emission transfer (FRET) effected by a complex system of displacing looped nucleic acids that are bound to a fluorescence emitter and a quencher to produce a fluorescent signal.
Richard, U.S. Pat. No. 5,645,987 describes an isothermal polymerization reaction which may be used to amplify the signal generated by a catalytic primer substrate and the products are detected by various methods. Western, et al., U.S. Pat. No. 6,110,677 describe a different method of amplifying an initial oligonucleotide target for use in diagnostic methods. Seeman, et al., U.S. 2006/0078910 describe formations of nucleic acid nanostructures from crossover domains.
Levy, M., et al., Proc. Natl. Acad. Sci. USA (2003) 100:6416-6421 describe a cross-catalytic cleavage method of amplification, but does not suggest its incorporation into assay methods. Lizardi, et al., U.S. Pat. No. 5,118,801 describe a signal amplification method which involves taking advantage of allosteric changes and employs conventional amplification methods and conventional detection using fluorescence and calorimetric methods. Liu, et al., U.S. Pat. No. 5,914,230 describe an alternative method of amplifying an initial nucleic acid signal and detection by means of a label.
Kool, U.S. Pat. No. 5,714,320 describes rolling circular amplification which is used in the present invention to generate signal amplification polymers.
The present invention provides particularly effective assay approaches which result in amplified signals permitting small amounts of analyte to be detected using convenient readouts requiring no instrumentation.